A living cell is a complex assembly of molecular elements; to function properly, its constituent molecules must form associations and operate in an organized manner. Certain components bind together to form molecular superstructures, including organelles which compartmentalize cellular activities and filaments which impart order and control motility. Other components exist in soluble form, and may move freely throughout the cell or, alternatively, within a subcellular compartment.
Cells are also equipped with elements that synthesize, process, and secrete a designated subset of proteins. This so-called secretory pathway includes membrane associated structures, such as the endoplasmic reticulum and Golgi apparatus, as well as a number of resident soluble molecules which participate in the processing of secreted proteins. Proteins which are to be secreted pass through the Golgi apparatus, where they are packaged for export from the cell. Accompanying them, by virtue of the continual vesicular transport of membrane and endoplasmic reticulum luminal contents, are soluble proteins properly residing in the endoplasmic reticulum.
To avoid continuously losing and needing to resynthesize these resident proteins, the cell uses a membrane-bound receptor localized in or near the Golgi apparatus for their retrieval (Lewis and Pelham, 1992, Cell 68:353-364). The receptor binds to a specific carboxy-terminal amino acid sequence which serves as a marker of what proteins are to be returned to the endoplasmic reticulum; this sequence is generally lysine-aspartic acid-glutamic acid-leucine (Lys-Asp-Glu-Leu in the three-letter amino acid code, KDEL in the single-letter code, referred to herein as "KDEL"), so that the receptor is generally referred to as the KDEL (SEQ ID NO:37) receptor (Munro and Pelham, 1987, Cell 48:899-907; Pelham, 1988, EMBO J. 7:913-918). The human KDEL receptor has been characterized as a seven-transmembrane domain protein which is a temporary resident of the Golgi apparatus: upon binding to a KDEL (SEQ ID NO:37)-containing ligand, it moves to the endoplasmic reticulum, where the ligand is released (Townsley et al., 1993, EMBO J. 12:2821-2829).
Among the molecules interacting with the KDEL receptor are certain members of a class of proteins, referred to as "heat shock proteins", which form associations with nascent polypeptides in the endoplasmic reticulum and act as molecular "chaperones", escorting a protein through the assembly process prior to its secretion (Frydman et al., 1994, Nature 370:111-117; Hendrick and Hartl, Annu. Rev. Biochem. 62:349-384; Hartl, 1996, Nature 381:571-580). Heat shock proteins constitute a highly conserved class of proteins selectively expressed in cells under stressful conditions, such as sudden increases in temperature or glucose deprivation. Able to bind to a wide variety of other proteins in their non-native state, heat shock proteins participate in the manufacture of these bound proteins, including their synthesis, folding, assembly, disassembly and translocation (Freeman and Morimoto, 1996, EMBO J. 15:2969-2979; Lindquist and Craig, 1988, Annu. Rev. Genet. 22:631-677; Hendrick and Hartl, 1993, Annu. Rev. Biochem. 62:349-384).
Two heat shock proteins which contain ligand sequences for the KDEL receptor are gp96 and BiP. Found in higher eukaryotes but not in Drosophila or yeast, gp96 appears to have evolved relatively recently, perhaps by a duplication of the gene encoding the cytosolic heat shock protein hsp90, to which it is highly related (Li and Srivastava, 1993, EMBO J. 12:3143-3151; identity between human hsp90 and murine gp96 is about 48 percent; Wiech et al., 1992, Nature 358:169-170; Melnick et al., 1992, J. Biol. Chem. 267:21303-21306; Melnick et al., 1994, Nature 370:373-375; Schaiff et al., 1992, J. Exp. Med. 176:657-666; Ramakrishnan et al., 1995, DNA and Cell Biol. 14:373-384). BiP (also referred to in the literature as grp78) forms a complex with newly synthesized immunoglobulin chains (Bole et al., 1986, J. Cell Biol. 102:1558-1566).
Under certain circumstances, it may be desirable to interfere with the normal control of KDEL (SEQ ID NO:37)-mediated protein redistribution. According to the present invention, a subject may benefit, for example, from the secretion of heat shock proteins which are normally retained in the endoplasmic reticulum but which have beneficial immunogenic effects when released.
Heat shock proteins are believed to play a role in the immune response in several contexts. Inoculation with heat shock protein prepared from tumors of experimental animals has been shown to induce immune responses in a tumor-specific manner; that is to say, heat shock protein gp96 purified from a particular tumor could induce an immune response which would inhibit the growth of cells from the identical tumor of origin, but not other tumors, regardless of relatedness (Srivastava and Maki, 1991, Curr. Topics Microbiol. 167:109-123). High-resolution gel electrophoresis has indicated that tumor-derived gp96 may be heterogeneous at the molecular level; evidence suggests that the source of this heterogeneity may be populations of small peptides adherent to the heat shock protein, which may number in the hundreds (Feldweg and Srivastava, 1995, Int. J. Cancer 63:310-314). Indeed, an antigenic peptide of vesicular stomatitis virus has been shown to associate with gp96 in virus infected cells (Nieland et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:6135-6139). It has been suggested that this accumulation of peptides is related to the localization of gp96 in the endoplasmic reticulum, where it may act as a peptide acceptor and accessory to peptide loading of major histocompatability complex class I molecules (Li and Srivastava, 1993, EMBO J. 12:3143-3151; Suto and Srivastava, 1995, Science 269:1585-1588). Recent studies have shown that protein disulfide isomerase ("PDI"), a resident luminal protein of the endoplasmic reticulum having a molecular weight of approximately 60kDa, may also function as a peptide acceptor (Lammert et al., 1997, Eur. J. Immunol. 27:1685-1690).
Further, the use of heat shock proteins as adjuvants to stimulate an immune response has been proposed (see, for example, Edgington, 1995, Bio/Technol. 13:1442-1444; PCT Application International Publication Number WO 94/29459 by the Whitehead Institute for Biomedical Research, Richard Young, inventor, and references infra). One of the best known adjuvants, Freund's complete adjuvant, contains a mixture of heat shock proteins derived from mycobacteria (the genus of the bacterium which causes tuberculosis); Freund's complete adjuvant has been used for years to boost the immune response to non-mycobacterial antigens. A number of references suggest, inter alia, the use of isolated mycobacterial heat shock proteins for a similar purpose, including vaccination against tuberculosis itself (Lukacs et al., 1993, J. Exp. Med. 178:343-348; Lowrie et al., 1994, Vaccine 12:1537-1540; Silva and Lowrie, 1994, Immunology 82:244-248; Lowrie et al., 1995, J. Cell. Biochem. Suppl. 0(19b):220; Retzlaff et al., 1994, Infect. Immun. 62:5689-5693; PCT Application International Publication No. WO 94/11513 by the Medical Research Council, Colston et al., inventors; PCT Application International Publication No. WO 93/1771 by Biocine Sclavo Spa, Rappuoli et al., inventors).
Increased levels of autologous heat shock proteins may also lead to an improved immune response by virtue of the association of heat shock proteins with endogenous antigenic peptides (International Application No. PCT/US96/13233 by Rothman et al.). Such activity is distinct from the traditionally utilized adjuvant activity of heterologous heat shock proteins.
The present invention is directed toward increasing the secretion of antigenic heat shock protein complexes by inhibiting KDEL receptor-mediated return of such complexes to the endoplasmic reticulum. Analogous methods may be used to increase the secretion of other proteins of interest which normally would tend to be retained via the KDEL receptor.